Process and reagents for processing synthetic oligonucleotides

• US 5,656,741 A
• Filed: 05/05/1995
• Issued: 08/12/1997
• Est. Priority Date: 03/30/1992
• Status: Expired due to Fees

• Current Assignee: Barrskogen, Inc.
• Original Assignee: Barrskogen, Inc.
• Inventors: Chow, Flora, Kempe, Tomas
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): Weiss, Bonnie D.
• Application Number:
  US08/435,526
• Time in Patent Office: 830 Days
• Field of Search: 536/22.1, 536/24.3, 536/24.31, 536/24.33, 536/25.3
• US Class Current: 536/25.3
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00306 Reactor vessels in a multiple arrangement
• CPC Class Codes: B01J 2219/00414 using suction
• CPC Class Codes: B01J 2219/00418 using pressure
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00731 Saccharides
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 40/12 Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 for creating libraries

Abstract

Reagents useful for the rapid processing of synthetic oligonucleotides, including a basic reagent for the cleavage and deprotection of the synthesized oligonucleotides from a support, and a precipitating reagent useful for recovering the synthesized oligonucleotide by precipitation from solution. The basic reagent optionally includes a wetting agent useful for the cleavage from lipophilic supports.

Claims

1. A process for the cleavage, deprotection, and recovery of a synthetic oligonucleotide comprising guanosine bases protected with base labile protecting groups selected from the group consisting of isobutyryl groups and phenoxyacetyl groups, wherein the oligonucleotide is synthesized on a solid support matrix, the process comprising the steps of:

(a) immersing the support in a basic reagent solution, comprising a sodium, potassium, calcium or lithium salt, of a type and concentration suitable to cause the cleavage of synthesized oligonucleotide from the support within about 5 minutes incubation at room temperature, and thereafter to cause the deprotection of isobutyryl groups within about 30 minutes and of phenoxyacetyl groups within about 10 minutes, at elevated temperatures up to 85°

,

b) incubating the solution for about 5 minutes or less under conditions suitable to cause the cleavage of substantially all synthesized oligonucleotide,

c) incubating the solution for about 30 minutes or less under conditions suitable to cause the deprotection of substantially all protecting groups from the cleaved oligonucleotide,

d) combining with the solution, at room temperature, a precipitating solvent reagent, comprising between about 0.5% and 5% acetic acid in an alcohol selected from the group consisting of 2-propanol, propanol, ethanol, butanol, and ethanolamine, suitable to precipitate the cleaved, deprotected oligonucleotide from the combined solution within about 10 minutes incubation at room temperature,

e) incubating the combined solution in order to precipitate substantially all cleaved, deprotected oligonucleotide within about 10 minutes.

2. A process according to claim 1 wherein the basic reagent comprises a solution of 0.1 molar to 2 molar sodium hydroxide.

3. A process according to claim 2 wherein the basic reagent further comprises a wetting agent.

4. A process according to claim 3 wherein the wetting agent comprises 2-propanol, present at a concentration of 10% in the acidic reagent solution, based on the volume of the solution.

5. A process according to claim 1 wherein the oligonucleotide has been synthesized by the use of phosphotriester nucleotides.

6. A process according to claim 1 wherein the oligonucleotide has been synthesized by the use of phosphoramidite nucleotides.

7. A process according to claim 1 wherein the cleaved, deprotected, precipitated oligonucleotide is present as a sodium salt.

8. A process according to claim 7 wherein the oligonucleotide is an antisense DNA sequence.

9. A process according to claim 1 wherein the oligonucleotide is a phosphorothioate DNA derivative.

10. A reagent kit useful for the cleavage, deprotection, and recovery of synthetic oligonucleotides protected with base labile protecting groups selected from the group consisting of isobutyryl groups and phenoxyacetyl groups and synthesized on a solid support matrix, the reagent kit comprising:

(a) a basic reagent solution, comprising a sodium, potassium, calcium or lithium salt, of a type and concentration suitable to cause the cleavage of synthesized oligonucleotide from the support within about 5 minutes incubation at room temperature, and thereafter to cause the deprotection of isobutyl groups within about 30 minutes incubation and of phenoxyacetyl groups within about 10 minutes incubation at elevated temperatures up to 85°

C., and

(b) a precipitating solvent reagent comprising between about 0.5% and 5% acetic acid in an alcohol selected from the group consisting of 2-propanol, propanol, ethanol, butanol, and ethanolamine suitable to precipitate the cleaved, deprotected oligonucleotide from the combined solution with about 10 minutes incubation at room temperature.

11. A reagent kit according to claim 10 wherein the basic reagent is a solution of 0.1 molar to 2 molar sodium hydroxide.